While the genetic factors responsible for age related macular degeneration (AMD) are becoming unraveled, the pathophysiology of the disease remains difficult to understand. AMD is characterized by an accumulation of drusen between the basal lamina of the retinal pigment epithelium (RPE) and Bruch's membrane resulting in progressive degeneration of RPE cells and photoreceptors. Understanding the cellular and molecular origins of drusen will help in finding new therapeutic targets to prevent or slow down the progression of the disease. The objective of this study is to determine the role of RPE cells in drusen formation. The underlying hypothesis is that RPE cells are involved in drusen formation and that an aberrant response of RPE from AMD donors to different environmental stressors is a cause of drusen accumulation. Protein expression patterns and especially proteins secreted by RPE cells, detectable by proteomic techniques, are expected to be a direct reflection of the relationship between RPE cells and drusen formation. Using a cutting edge proteomics approach (stable isotope labeling by amino acids) we show that RPE cells secrete a variety of proteins that have been reported to be major constituent of drusen. Additionally, our data reveals that RPE cells from AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-CFH variant) secreted 2 to 3 fold more of these proteins than RPE from age matched healthy donors. In Specific Aim I, we will determine differentially expressed proteins between AMD RPE and healthy RPE using quantitative proteome profiling. We will focus on secreted proteins but eventually other subcellular fraction will be analyzed for comprehensive comparative proteomics. In Specific Aim II, changes in protein secretion in AMD RPE cells versus healthy RPE cells will be monitored following oxidative and/or inflammatory mediated injury. Overall, the results will help identify abnormal protein expression and trafficking associated with AMD pathogenesis in the retinal pigment epithelial cells. We anticipate that the application of newly emerging proteomic techniques will result in an improved understanding of the pathways involved in AMD pathogenesis and will help in finding new therapeutic targets to delay or stop progression of the disease. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page Principal Investigator/Program Director (Last, First, Middle):Hathout, Yetrib The goal of this project is to study cellular and molecular mechanisms involved in age related macular degeneration (AMD) pathology. We will implement a proteomic approach to assess differential protein secretion in RPE cell cultures derived from AMD and age matched healthy donors.